M01028
AMINO ACID AND FATTY ACID PROFILLING FROM MODIFIED
SORGHUM FLOUR
Yohanes Martono, Vellisya Puspaningsih, Sri Hartini
Chemistry Department, Faculty of Science and Mathematics
Satya Wacana Christian University, Salatiga, Indonesia
E-mail: yohanes_mart@yahoo.co.id.
ABSTRACT
Sorghum is potential food source that can be developed in Indonesia. Sorghum has high content of
protein and rich in unsaturated fatty acid. The aim of this research was to identify and determine the
content of amino acid and fatty acid from modified shorgum flour. Shorgum was modified by
fermentation and fortification with peanuts. Identification of amino acid was done by using High
Performance Liquid Chromatography (HPLC)
while fatty acid was analysed by using Gas
Chromatography – Mass Spektrometer (GC-MS). Amino acid and fatty acid from modified sorghum
flour was compared with unmodified sorghum flour. Data were analysed descriptively. The results
showed that amino acid in modified sorghum flour were aspartic acid, glutamic acid, serine, histidine,
glysine, arginine, alanin, tyrosine, methionine, valine, phenylalanine, ileusine, leusin and lysine.The
highest content of amino acid was glutamic acid, 148.98 ppm and the lowest or limitted amino acid in
sorghum flour was methionine, 19.95 ppm. The main compound unsaturated fatty acid of modified
sorghum flour were oleic acid and linoleic acid.The contents of both main unsaturated fatty acids were
35,64% and 32,41% respectively.
Key words: amino acid, fatty acid, sorghum, modified flour.
INTRODUCTION
One of the potential crop used as a functional food is sorghum. [24] states that the
chemical, sorghum is an important potential source of functional food (nutraceuticals
phenolic). This plant also has great potential to be developed commercially in Indonesia
particularly dry area [18], because it is supported by ecological condition [19].
Problems faced currently is limited in sorghum cultivation for lack of information
about the potential of sorghum. Over the years, sorghum only be used as animal feed
[10][11]. According [19], one of factors that caused this limited was the high rate of antinutritional substances, tannins (about 0.40 to 3.60%) contained in sorghum. It is therefore
necessary optimizations in the reduction of anti-nutritional tannins compounds.
Sorghum has a high protein content (10.11%) [19], but still lower in amino acid
variations and causes its low protein digestibility [3]. In fact, the content of amino acids of
protein determine the nutritional value of food [19]. Therefore, the necessary modifications
through fortification of peanut (Arachis hypogaea). Peanuts not only have high levels of
protein (25.3 g in 100 g) but also contains essential amino acids that can not be produced by
the human body. Besides, unsaturated fatty acids of peanuts is high (42.8 g in 100 g) [25].
Food containing high protein may not have a good protein quality. Protein quality of a
food is determined composition and availability of amino acids that can be absorbed by the
body [6][1].
It is necessary to identify amino acids of protein beside determine protein
content in food. The aim of this research was to identify and determine the content of amino
acid and fatty acid from modified shorgum flour. Shorgum was modified by fermentation and
fortification with peanuts.
METHODS
Materials
The sample used is a sorghum (Sorghum bicolor L.) varieties derived from Surakarta,
peanuts (Arachis hypogaea) obtained from Kulon Progo in Ambarawa market, and tempeh
fermipan Raprima brand. All chemicals were pro-analysis grade and were purchased from
Merck, Germany. Amino acids standard were purchased from Sigma Aldrich.
Instrumentations
Instruments used in analysis were spektrophotometer (Optizen UV 2120, Korea),
waterbath (Memmert WNB 14, Germany), centrifuge (EBA 21
Hettich Zentrifugen,
Germany), mass balanced (Mettler H-80, Germany), soxhlet, rotary evaporator (Buchi R114, Switzerland), HPLC (Shimadzu LC 10, Japan), GCMS (Shimadzu QP2010S, Japan).
Procedure
Degradation of Tannins Content in Sorghum Seed [26].
Decreased levels of tannins in sorghum was done by immersion method in 0.3% Na 2CO3 for
8 hours, followed by immersion in 0.2% Na 2HPO4 for 2 hours. Method performed with 1:3
ratio (sorghum seed: solvent).
Content Determination of Tannins in Sorghum Seed by Spectrophotometric
Three mL sample solution were poured into 25 ml volumetric flask. Solution was
added by 1 mL 0.016 M K3Fe(CN)3 and 1 mL 0.012 M FeCl3. Solution was shaken and
incubated for 15 min. Then, solutions was added by 3 ml 6.03 M H 3PO4 . Solution was
shaken and incubated for 2 minutes. Then, the solution was added 2 ml of gelatin.
Absorbance of the solution was measured at 718 nm. Tannic acid was used as external
standard.
Optimization of Fortification and Fermentation Condition Based on Sorghum Soluble
Protein Content
Sorghum seeds were boiled for 30 minutes and then cooled. Then, sorghum seeds
were inoculated with the microbes, Rhizopus oligosporus 2.5% (w / w), 5% (w / w) and 10%
(w / w) and fortified with peanuts (Arachis hypogaea) in various concentrations 0%, 2.5%,
5%, 7.5%, 10% (w / w). Each was fermented with the time variation of 0 hours to 48 hours
with an analysis time per 12 hours. The results then dried, pulverized and sifted. Soluble
protein content was measured by Biuret method.
Analysis of Protein Content in Sorghum Seed by Biuret Method
One gram samples was added by 9 ml of distilled water and then added 1 ml 1 M
NaOH. Then, the solution was heated in a water bath with a temperature of 90 ° C and
cooled. Solutions was centrifuged on 3000 rpm for 10 minutes. 1 ml supernatant was taken
and added by 4 ml Biuret reagent (0.15 g + 0.6 g KNaTartaric CuSO4.5H2O in 50 mL
volumetric flask and added 30 ml of 10% NaOH and fulfilled with distilled water in a 100 ml
flask). Solution was incubated for 30 min at room temperature. Absorbance of the solution
was measured with a spectrophotometer at 550 nm. Protein solution (BSA) was used as a
standard with various concentrations.
Hydrolysis Of Proteins
Protein hydrolysis using a reflux, which is 15 g of sample was added 70 ml 7.5 M HCl
and heated in water bath with a temperature of 90 ° C. Hydrolyzed samples were filtered and
the filtrate evaporated at a temperature of 90 °C to remove HCl.
Amino Acid Analysis
60 mg sample was added by 4 ml 6 N HCl and heated for 24 hours at 110 oC.
Subsequently, the solution was neutralized (pH = 7) with 6 N NaOH and filtered with
Whatman filter paper 0.2 µm. Then, 30 mL of sample solution was derivated by 300 mL OPA
(o-phthalaldehyde) and stirred for 5 minutes. Then, 20 mL solution was injected into the
HPLC injector.
Chromatography Conditions of HPLC
Identification of amino acids using HPLC method on the condition of stationary phase
column Licrospher 100 RP 18 (125 x 4.0 mm, 5 m). The elution was isocratic system with
the mobile phase solvent A = methanol : 50 mM sodium acetate: THF (2 : 96 : 2 v/v/v) pH
6.8 and solvent B = 65% methanol. Flow rate used was 1.5 ml / min and detected using a
Flourescence detector (420 E. WATERS)
Fatty Acid Extraction
10 g samples were grinded put in Soxhlet extraction tube, then the water flows
through the condenser. Solvent extraction tube fitted with enough diethyl ether for 4 hours.
Then, again continued for 2 hours with the same solvent. Weigh heavy residue on the bottle
net weight expressed as fats and oils.
Identification of Unsaturated Fatty Acids by GC-MS Method
Identify the building blocks of sorghum seed fatty acids were analyzed using Gas
Chromatography Mass Spectrophotometer (Shimadzu QP2010S), in the Laboratory of
Organic Chemistry, Faculty of Science, University of Gadjah Mada in operational conditions:
Column
: EGILENT J&W DB-5 (30 m x 0,25 mm)
Carier gas
: Helium
Temperature gradien : 70oC for 5 minutes and 10oC/min-300oC
ionization
: Electron Impact (EI)
RESULTS AND DISCUSSION
Degradation of Tannins Content in Sorghum Seed
Quantitative assay of tannins in sorghum grains showed that the method used has
significant impact on tannin degradation. Figure 1 shows that the levels of tannins in
sorghum is still high at 0.08% (w / w), these results are consistent with studies [10][21] which
mentioned that the phenol content in sorghum was still high and dominated by tannin.
Tannins contained in the condensed tannin sorghum is known as proantosianin.
Proantosianin are polymers of flavanoid-containing unit or flavan 3-ol catechin. These
compounds include polyphenolic compounds that can form complexes with proteins resulting
in lower quality and protein digestibility [24][26]. Tannins have the ability to precipitate
proteins into complexes because of its ability to make copolymer crosslinking protein. Bonds
between protein complexes with tannin sorghum causing protein is difficult to be digested or
inhibit digestive enzymes work processes [26]
Soaking using salt solution capable of causing tannins bind to proteins (especially
albumin and globumin) and become soluble. This is because albumin is a water-soluble
protein and globulin proteins that are soluble in salt solution [26]. This is why the methods
used are quite effective in reducing the tannin in sorghum (Table 1).
Table 1. Data percent degradation (%) of tanin content
Method
Control
I
Initial (%)
0.081
0.081
Final (%)
0.081
0.009
% Degradation
88.88
Note : Method I : Soaked by Na2CO3 and followed by Na2HPO4
Tannin Content (%)
0,1
0,081
0,08
0,06
0,04
0,009
0,02
0
initial
Method I
Figure 1. Degradation tannin content by method used
Optimization of Protein Content in Modified Sorghum Seed
Results soluble protein content in sorghum that have not been processed and
fermented reached 12.78%, but when processed or cooked dissolved protein levels
decreased to 0.58% (Figure 2) Sorghum is a food that cannot be eaten, the need for
processing to be consumed by humans. However, sorghum grain processing (boiling
process) lead to lower levels of protein and protein digestibility in sorghum. This happens
Soluble Protein Content
(%)
because protein polymers will be denatured enzimatically.
15
10
5
0
Sorghum
boiled
sorghum
peanuts
Figure 2. Soluble protein content of initial sorghum, boiled sorghum and peanuts.
Fortification by mixing two or more different sources of protein that have different
limiting amino acids will be complementary of protein content [25]. Soluble protein content of
peanuts is high at 7.92%. In other words, peanuts can be used as another source of protein
that is able to improve the nutrition and quality of food.
Optimization conditions of Peanut Fortification and Sorghum Fermentation
Optimization of conditions fortification followed by a review of the interaction of each
variable, ie Rhyzopus oligosporus microbial levels, the addition of peanuts and fermentation
time. Results of mathematical modeling interpolation method with matlab 6.5 programs
produce fortification and fermentation conditions are optimal in terms of the levels of soluble
protein content of microbes is 2.5% with the addition of peanuts by 9% and fermented for 49
hours (Figure 3).
Figure 3. Mathematical modeling interpolation method with matlab 6.5 programs on sorghum
flour modification
Based on previous research showed that the protein at the optimum point relatively
high in protein. High levels of this protein may reduce the quality of the flour so that the
expanding capacity of flour into decline. Therefore, the optimal conditions for sorghum
modification is based on the interaction between variables microbial concentration, peanuts
concentration and fermentation time that provide soluble protein content of at least 6% (total
protein > 7% according to the terms of Indonesia National Standadr (SNI 01-3751-2006)
wheat flour quality requirements). The statistical analysis (P
SORGHUM FLOUR
Yohanes Martono, Vellisya Puspaningsih, Sri Hartini
Chemistry Department, Faculty of Science and Mathematics
Satya Wacana Christian University, Salatiga, Indonesia
E-mail: yohanes_mart@yahoo.co.id.
ABSTRACT
Sorghum is potential food source that can be developed in Indonesia. Sorghum has high content of
protein and rich in unsaturated fatty acid. The aim of this research was to identify and determine the
content of amino acid and fatty acid from modified shorgum flour. Shorgum was modified by
fermentation and fortification with peanuts. Identification of amino acid was done by using High
Performance Liquid Chromatography (HPLC)
while fatty acid was analysed by using Gas
Chromatography – Mass Spektrometer (GC-MS). Amino acid and fatty acid from modified sorghum
flour was compared with unmodified sorghum flour. Data were analysed descriptively. The results
showed that amino acid in modified sorghum flour were aspartic acid, glutamic acid, serine, histidine,
glysine, arginine, alanin, tyrosine, methionine, valine, phenylalanine, ileusine, leusin and lysine.The
highest content of amino acid was glutamic acid, 148.98 ppm and the lowest or limitted amino acid in
sorghum flour was methionine, 19.95 ppm. The main compound unsaturated fatty acid of modified
sorghum flour were oleic acid and linoleic acid.The contents of both main unsaturated fatty acids were
35,64% and 32,41% respectively.
Key words: amino acid, fatty acid, sorghum, modified flour.
INTRODUCTION
One of the potential crop used as a functional food is sorghum. [24] states that the
chemical, sorghum is an important potential source of functional food (nutraceuticals
phenolic). This plant also has great potential to be developed commercially in Indonesia
particularly dry area [18], because it is supported by ecological condition [19].
Problems faced currently is limited in sorghum cultivation for lack of information
about the potential of sorghum. Over the years, sorghum only be used as animal feed
[10][11]. According [19], one of factors that caused this limited was the high rate of antinutritional substances, tannins (about 0.40 to 3.60%) contained in sorghum. It is therefore
necessary optimizations in the reduction of anti-nutritional tannins compounds.
Sorghum has a high protein content (10.11%) [19], but still lower in amino acid
variations and causes its low protein digestibility [3]. In fact, the content of amino acids of
protein determine the nutritional value of food [19]. Therefore, the necessary modifications
through fortification of peanut (Arachis hypogaea). Peanuts not only have high levels of
protein (25.3 g in 100 g) but also contains essential amino acids that can not be produced by
the human body. Besides, unsaturated fatty acids of peanuts is high (42.8 g in 100 g) [25].
Food containing high protein may not have a good protein quality. Protein quality of a
food is determined composition and availability of amino acids that can be absorbed by the
body [6][1].
It is necessary to identify amino acids of protein beside determine protein
content in food. The aim of this research was to identify and determine the content of amino
acid and fatty acid from modified shorgum flour. Shorgum was modified by fermentation and
fortification with peanuts.
METHODS
Materials
The sample used is a sorghum (Sorghum bicolor L.) varieties derived from Surakarta,
peanuts (Arachis hypogaea) obtained from Kulon Progo in Ambarawa market, and tempeh
fermipan Raprima brand. All chemicals were pro-analysis grade and were purchased from
Merck, Germany. Amino acids standard were purchased from Sigma Aldrich.
Instrumentations
Instruments used in analysis were spektrophotometer (Optizen UV 2120, Korea),
waterbath (Memmert WNB 14, Germany), centrifuge (EBA 21
Hettich Zentrifugen,
Germany), mass balanced (Mettler H-80, Germany), soxhlet, rotary evaporator (Buchi R114, Switzerland), HPLC (Shimadzu LC 10, Japan), GCMS (Shimadzu QP2010S, Japan).
Procedure
Degradation of Tannins Content in Sorghum Seed [26].
Decreased levels of tannins in sorghum was done by immersion method in 0.3% Na 2CO3 for
8 hours, followed by immersion in 0.2% Na 2HPO4 for 2 hours. Method performed with 1:3
ratio (sorghum seed: solvent).
Content Determination of Tannins in Sorghum Seed by Spectrophotometric
Three mL sample solution were poured into 25 ml volumetric flask. Solution was
added by 1 mL 0.016 M K3Fe(CN)3 and 1 mL 0.012 M FeCl3. Solution was shaken and
incubated for 15 min. Then, solutions was added by 3 ml 6.03 M H 3PO4 . Solution was
shaken and incubated for 2 minutes. Then, the solution was added 2 ml of gelatin.
Absorbance of the solution was measured at 718 nm. Tannic acid was used as external
standard.
Optimization of Fortification and Fermentation Condition Based on Sorghum Soluble
Protein Content
Sorghum seeds were boiled for 30 minutes and then cooled. Then, sorghum seeds
were inoculated with the microbes, Rhizopus oligosporus 2.5% (w / w), 5% (w / w) and 10%
(w / w) and fortified with peanuts (Arachis hypogaea) in various concentrations 0%, 2.5%,
5%, 7.5%, 10% (w / w). Each was fermented with the time variation of 0 hours to 48 hours
with an analysis time per 12 hours. The results then dried, pulverized and sifted. Soluble
protein content was measured by Biuret method.
Analysis of Protein Content in Sorghum Seed by Biuret Method
One gram samples was added by 9 ml of distilled water and then added 1 ml 1 M
NaOH. Then, the solution was heated in a water bath with a temperature of 90 ° C and
cooled. Solutions was centrifuged on 3000 rpm for 10 minutes. 1 ml supernatant was taken
and added by 4 ml Biuret reagent (0.15 g + 0.6 g KNaTartaric CuSO4.5H2O in 50 mL
volumetric flask and added 30 ml of 10% NaOH and fulfilled with distilled water in a 100 ml
flask). Solution was incubated for 30 min at room temperature. Absorbance of the solution
was measured with a spectrophotometer at 550 nm. Protein solution (BSA) was used as a
standard with various concentrations.
Hydrolysis Of Proteins
Protein hydrolysis using a reflux, which is 15 g of sample was added 70 ml 7.5 M HCl
and heated in water bath with a temperature of 90 ° C. Hydrolyzed samples were filtered and
the filtrate evaporated at a temperature of 90 °C to remove HCl.
Amino Acid Analysis
60 mg sample was added by 4 ml 6 N HCl and heated for 24 hours at 110 oC.
Subsequently, the solution was neutralized (pH = 7) with 6 N NaOH and filtered with
Whatman filter paper 0.2 µm. Then, 30 mL of sample solution was derivated by 300 mL OPA
(o-phthalaldehyde) and stirred for 5 minutes. Then, 20 mL solution was injected into the
HPLC injector.
Chromatography Conditions of HPLC
Identification of amino acids using HPLC method on the condition of stationary phase
column Licrospher 100 RP 18 (125 x 4.0 mm, 5 m). The elution was isocratic system with
the mobile phase solvent A = methanol : 50 mM sodium acetate: THF (2 : 96 : 2 v/v/v) pH
6.8 and solvent B = 65% methanol. Flow rate used was 1.5 ml / min and detected using a
Flourescence detector (420 E. WATERS)
Fatty Acid Extraction
10 g samples were grinded put in Soxhlet extraction tube, then the water flows
through the condenser. Solvent extraction tube fitted with enough diethyl ether for 4 hours.
Then, again continued for 2 hours with the same solvent. Weigh heavy residue on the bottle
net weight expressed as fats and oils.
Identification of Unsaturated Fatty Acids by GC-MS Method
Identify the building blocks of sorghum seed fatty acids were analyzed using Gas
Chromatography Mass Spectrophotometer (Shimadzu QP2010S), in the Laboratory of
Organic Chemistry, Faculty of Science, University of Gadjah Mada in operational conditions:
Column
: EGILENT J&W DB-5 (30 m x 0,25 mm)
Carier gas
: Helium
Temperature gradien : 70oC for 5 minutes and 10oC/min-300oC
ionization
: Electron Impact (EI)
RESULTS AND DISCUSSION
Degradation of Tannins Content in Sorghum Seed
Quantitative assay of tannins in sorghum grains showed that the method used has
significant impact on tannin degradation. Figure 1 shows that the levels of tannins in
sorghum is still high at 0.08% (w / w), these results are consistent with studies [10][21] which
mentioned that the phenol content in sorghum was still high and dominated by tannin.
Tannins contained in the condensed tannin sorghum is known as proantosianin.
Proantosianin are polymers of flavanoid-containing unit or flavan 3-ol catechin. These
compounds include polyphenolic compounds that can form complexes with proteins resulting
in lower quality and protein digestibility [24][26]. Tannins have the ability to precipitate
proteins into complexes because of its ability to make copolymer crosslinking protein. Bonds
between protein complexes with tannin sorghum causing protein is difficult to be digested or
inhibit digestive enzymes work processes [26]
Soaking using salt solution capable of causing tannins bind to proteins (especially
albumin and globumin) and become soluble. This is because albumin is a water-soluble
protein and globulin proteins that are soluble in salt solution [26]. This is why the methods
used are quite effective in reducing the tannin in sorghum (Table 1).
Table 1. Data percent degradation (%) of tanin content
Method
Control
I
Initial (%)
0.081
0.081
Final (%)
0.081
0.009
% Degradation
88.88
Note : Method I : Soaked by Na2CO3 and followed by Na2HPO4
Tannin Content (%)
0,1
0,081
0,08
0,06
0,04
0,009
0,02
0
initial
Method I
Figure 1. Degradation tannin content by method used
Optimization of Protein Content in Modified Sorghum Seed
Results soluble protein content in sorghum that have not been processed and
fermented reached 12.78%, but when processed or cooked dissolved protein levels
decreased to 0.58% (Figure 2) Sorghum is a food that cannot be eaten, the need for
processing to be consumed by humans. However, sorghum grain processing (boiling
process) lead to lower levels of protein and protein digestibility in sorghum. This happens
Soluble Protein Content
(%)
because protein polymers will be denatured enzimatically.
15
10
5
0
Sorghum
boiled
sorghum
peanuts
Figure 2. Soluble protein content of initial sorghum, boiled sorghum and peanuts.
Fortification by mixing two or more different sources of protein that have different
limiting amino acids will be complementary of protein content [25]. Soluble protein content of
peanuts is high at 7.92%. In other words, peanuts can be used as another source of protein
that is able to improve the nutrition and quality of food.
Optimization conditions of Peanut Fortification and Sorghum Fermentation
Optimization of conditions fortification followed by a review of the interaction of each
variable, ie Rhyzopus oligosporus microbial levels, the addition of peanuts and fermentation
time. Results of mathematical modeling interpolation method with matlab 6.5 programs
produce fortification and fermentation conditions are optimal in terms of the levels of soluble
protein content of microbes is 2.5% with the addition of peanuts by 9% and fermented for 49
hours (Figure 3).
Figure 3. Mathematical modeling interpolation method with matlab 6.5 programs on sorghum
flour modification
Based on previous research showed that the protein at the optimum point relatively
high in protein. High levels of this protein may reduce the quality of the flour so that the
expanding capacity of flour into decline. Therefore, the optimal conditions for sorghum
modification is based on the interaction between variables microbial concentration, peanuts
concentration and fermentation time that provide soluble protein content of at least 6% (total
protein > 7% according to the terms of Indonesia National Standadr (SNI 01-3751-2006)
wheat flour quality requirements). The statistical analysis (P